This invention relates to the separation of molecules on polymer gels, in particular to the preparation of novel crosslinked polymer gels, the separation of molecules by techniques such as electrophoresis using these gels, novel crosslinking agents useful in the preparation of the gels, and novel intermediates useful in the synthesis of the crosslinking agents. The invention is especially suitable for electrophoretic applications and accordingly, for convenience, the invention will be further described with reference to electrophoresis. It is to be understood, however, that the gels, processes and crosslinking agents of the present invention are not so limited.
Polyacrylamide gel electrophoresis is an analytical technique whereby fragments of biomolecules, such as DNA, enzymes and proteins, may be separated and identified on the basis of their molecular size, weight and charge. Commercially available electrophoresis gels have conventionally been produced by copolymerisation of acrylamide with the symmetrical crosslinking agent, N,Nxe2x80x2-methylene bisacrylamide, otherwise known as BIS. Since both double bonds of BIS are of the same type their reactivities are essentially the same. Other known crosslinking agents include ethylene glycol diacrylate, dihydroxy ethylene-bisacrylamide (DHEBA), N,Nxe2x80x2-propylenebisacrylamide, diacrylamide dimethylether, 1,2-diacrylamide ethyleneglycol, ethyleneureabisacrylamide, N,Nxe2x80x2-bisacrylylcystamine and bisacrylamide methylether (BAME). As for BIS, the double bonds of these crosslinking agents are of the same type.
It has now been found that electrophoresis gels having surprisingly improved separating ability can be prepared using particular asymmetrical crosslinking agents.
Accordingly the invention provides a crosslinked polymer gel comprising a crosslinking moiety of the formula: 
wherein X and Xxe2x80x2 are independently selected from the group consisting of xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94 and xe2x80x94NRxe2x80x94, where R is H, alkyl or cycloalkyl,
R2 is a C1-C4 alkyl group,
Y is an optionally substituted non-aromatic divalent linking group, and
Z is O or S.
Preferably R2 is CH3.
The monomer or monomers used to prepare the gel may be any suitable monomer.
The crosslinked polymer gel may be prepared from monomers having the formula H2Cxe2x95x90CR5xe2x80x94COxe2x80x94NR3R4 where R3, R4 and R5 are each independently H or alkyl optionally monosubstituted by, for example, OH or C(O)CH2C(O) CH3. Examples of monomers include acrylamide, acrylamide derivatives or acrylamide substitutes known to the art such as N,N-dimethylacrylamide, methacrylamide, methyloylacrylamide, propylacrylamide, dipropyl acrylamide, isopropyl acrylamide, diisopropyl acrylamide, lactyl acrylamide, methoxyacrylamide and mixtures thereof. Preferably the monomer is acrylamide.
The linking group may be any suitable non-aromatic hydrocarbyl group, optionally including one or more heteroatoms selected from O, S, N and P.
Preferably X and Xxe2x80x2 are the same. Preferably Z is oxygen.
In another aspect of the invention there is provided a method of preparing a crosslinked polymer gel, said method including the step of subjecting one or more monomers to crosslinking polymerisation with one or more crosslinking agents of the formula I: 
wherein X and Xxe2x80x2 are independently selected from the group consisting of xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94 and xe2x80x94NRxe2x80x94, where R is H, alkyl or cycloalkyl,
R2 is a C1-C4 alkyl group,
Y is an optionally substituted non aromatic divalent linking group, and
Z is O or S.
R2 is preferably CH3.
The polymer gels according to the present invention may be prepared using one or more crosslinking agents of formula I, optionally in the presence of one or more conventional crosslinking agents known to the art. Preferably the crosslinking agent(s) is/are selected to provide a gel which is substantially transparent to visible light. Preferably the gel is an aqueous gel.
The polymer gels according to the present invention are useful for separating molecules, especially charged species, or species capable of bearing a charge such as biomolecules.
The polymer gel may be an electrophoretic gel. The electrophoretic gel may have a porosity gradient suitable for gradient gel electrophoresis. See for example, Polyacrylamide Gel Electrophoresis across a Molecular Sieve Gradient Margolis, J., Kenrick, K. G., Nature, 214, 1967, p1334-1336; Polyacrylamide Gel Electrophoresis in a Continuous Molecular Sieve Gradient, Margolis, J., Kenrick, K. G., Analytical biochemistry, 25, 1968, p347-362; and Practical System for Polyacrylamide Gradient Gel electrophoresis, Margolis, J., Laboratory Practice, 22, p107-109, 1973, the disclosures of which are incorporated herein by reference.
In a further aspect the invention provides a method of separating molecules comprising:
providing a crosslinked polymer gel by combining one or more monomers with a crosslinking agent of the formula I: 
wherein X and Xxe2x80x2 are independently selected from the group consisting of xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94 and xe2x80x94NRxe2x80x94, where R is H, alkyl or cycloalkyl,
R2 is a C1-C4 alkyl group, and
Y is an optionally substituted non aromatic divalent linking group, and
Z is O or S, optionally in the presence of an initiator, subjecting the monomer solution to polymerisation and crosslinking, placing a sample containing the molecules to be separated onto the gel, and subjecting the gel and sample to a separation technique. Preferably the separation technique is electrophoresis. The electrophoresis technique employed may be any of those known to the art, including one-, two- and multi-dimensional techniques. The electrophoresis technique may be gradient gel electrophoresis.
Preferably polymerisation is carried out on a solution of the monomer or monomers with the crosslinking agent.
The linking group is preferably selected to provide a crosslinking agent which is soluble in the monomer solution. For most applications involving acrylamide the solvent will be water, and accordingly it is preferred that the linking group is selected to provide a crosslinking agent which is soluble in water or water/acrylamide. Other solvents include DMF, THF, alcohols and other water miscible systems.
Where the solvent is water and/or the monomer is acrylamide, the hydrophilic/lipophilic balance of the linking group may be controlled so that the cross-linking agent is soluble in water or water/acrylamide. Accordingly if the linking group contains a large number of carbon atoms (eg. more than about 7) the effect on solubility can be offset by including sufficient oxygen atoms or other polar groups to provide a crosslinking agent which is soluble in the acrylamide/water solution.
Examples of divalent linking groups include alkylene, oxyalkylene, polyoxyalkylene, cycloalkylene, alkanedioyl, alkylenedisulphonyl, alkylenecarbonyl, thioalkylene, ureylene, oxalyl, aminoalkylene, alkylenedisulphonyl, heterocyclyl and groups of the formula xe2x80x94(R1)mxe2x80x94R2xe2x80x94(R3)nxe2x80x94, where R1 and R3 are selected from alkylene, cycloalkylene, heterocyclyl, oxyalkylene, polyoxyalkylene, alkylenecycloalkylene and alkyleneheterocyclyl; R2 is selected from a direct bond, xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94Sxe2x80x94Sxe2x80x94, alkylene, alkanedioyl, alkylenedioxy, alkylenedisulphonyl, xe2x80x94NRxe2x80x94, xe2x80x94NRC(O)Oxe2x80x94, xe2x80x94NRxe2x80x94C(O)xe2x80x94NRxe2x80x94, xe2x80x94NRC(O)xe2x80x94, xe2x80x94Nxe2x95x90Nxe2x80x94, xe2x80x94NRC(O)C(O)xe2x80x94NRxe2x80x94, xe2x80x94C(O)xe2x80x94, xe2x80x94C(S)xe2x80x94 and xe2x80x94RNNRxe2x80x94, where R is H, alkyl or cycloalkyl; m and n are 0 or 1 provided that m+nxe2x89xa00.
As used herein the term xe2x80x9cnon-aromatic hydrocarbyl groupxe2x80x9d means any divalent group comprising carbon and hydrogen which does not include an aromatic or heteroaromatic ring.
As used herein the term xe2x80x9calkylenexe2x80x9d, used either alone or in compound words such as xe2x80x9coxyalkylenexe2x80x9d, xe2x80x9ccarbonylalkylenexe2x80x9d denotes straight chain and branched C1-10 alkylene groups. Examples include methylene, ethylene, propylene, isopropylene, butylene, isobutylene, sec-butylene, pentylene, isopentylene, sec-pentylene, 1,2-dimethylpropylene, 1,1-dimethylpropylene, hexylene, 4-methylpentylene, 1-methylpentylene, 3-methylpentylene. 1,1-dimethylbutylene, 2,2-dimethylbutylene, 3,3-dimethylbutylene, 1,2-dimethylbutylene, 1,3-dimethylbutylene, 1,2,2-trimethylpropylene, 1,1,2-trimethylpropylene, heptylene, 5-methylhexylene, 1-methylhexylene, 2,2-dimethylpentylene, 3,3-dimethylpentylene, 4,4-dimethylpentylene, 1,2-dimethylpentylene, 1,3-dimethylpentylene, 1,4-dimethylpentylene, 1,2,3-trimethylbutyl, 1,1,2-trimethylbutylene and the like.
The term xe2x80x9ccycloalkylenexe2x80x9d, used alone or in compound words such as xe2x80x9calkylenecycloalkylenexe2x80x9d denotes divalent cyclic C3-7 alkyl groups. Examples include cyclopropyl, cyclobutyl, cyclopentyl and cycloheptyl.
The term xe2x80x9cheterocyclylxe2x80x9d as used alone or in compound names such as xe2x80x9calkyleneheterocyclylxe2x80x9d denotes 5 or 6 membered heterocyclic rings. Examples of 5 or 6 membered heterocyclic rings include pyrrolidine, imidazolidine, pyrazolidine, thiazolidine, isothiazolidine, oxazolidine, piperidine and piperazine.
In this specification the term xe2x80x9coptionally substitutedxe2x80x9d means that a group may or may not be further substituted with one or more groups selected from alkyl, cycloalkyl, alkenyl, alkynyl, halo, haloalkyl, haloalkynyl, hydroxy, alkoxy, alkenyloxy, haloalkoxy, haloalkenyloxy, nitro, amino, nitroalkyl, nitroalkenyl, nitroalkynyl, nitroheterocyclyl, alkylamino, dialkylamino, alkenylamine, alkynylamino, acyl, alkenacyl, alkynylacyl, acylamino, diacylamino, acyloxy, alkylsulphonyloxy, heterocyclyl, heterocycloxy, heterocyclamino, haloheterocyclyl, alkylsulphenyl, carboalkoxy, alkylthio, acylthio, phosphorous-containing groups such as phosphono and phosphinyl, and groups of the formula 
where X, Xxe2x80x2 and Z are as defined above.
The term xe2x80x9calkylxe2x80x9d, used either alone or in compound words such as xe2x80x9chaloalkylxe2x80x9d or xe2x80x9calkylthioxe2x80x9d, denotes straight chain or branched C1-6 alkyl groups. Examples include methyl, ethyl, propyl, isopropyl and the like.
The term xe2x80x9calkoxyxe2x80x9d denotes straight chain or branched alkoxy. preferably C1-10 alkoxy. Examples include methoxy, ethoxy, n-propoxy, isopropoxy and the different butoxy isomers.
The term xe2x80x9calkenylxe2x80x9d denotes groups formed from straight chain, branched or mono or polycyclic alkenes including ethylenically mono- or poly-unsaturated alkyl or cycloalkyl groups as previously defined, preferably C2-10 alkenyl. Examples of alkenyl include vinyl, allyl, 1-methylvinyl, butenyl, iso-butenyl, 3-methyl-2-butenyl, 1-pentenyl, cyclopentenyl, 1-methyl-cyclopentenyl, 1-hexenyl, 3-hexenyl, cyclohexenyl, 1-heptenyl, 3-heptenyl, 1-octenyl, cyclooctenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, 1-decenyl, 3-decenyl, 1,3-butadienyl, 1-4,pentadienyl, 1,3-cyclopentadienyl, 1,3-hexadienyl, 1,4-hexadienyl, 1,3-cyclohexadienyl, 1,4-cyclohexadienyl, 1,3-cycloheptadienyl, 1,3,5-cycloheptatrienyl, 1,3,5,7-cyclooctatetraenyl.
The term xe2x80x9chalogenxe2x80x9d denotes fluorine, chlorine, bromine or iodine, preferably chlorine or fluorine.
The term xe2x80x9cacylxe2x80x9d used either alone or in compound words such as xe2x80x9cacyloxyxe2x80x9d, xe2x80x9cacylthioxe2x80x9d, xe2x80x9cacylaminoxe2x80x9d or diacylaminoxe2x80x9d denotes carbamoyl, aliphatic acyl group and acyl group containing a heterocyclic ring which is referred to as heterocyclic acyl, preferably C1-10 acyl. Examples of acyl include carbamoyl; straight chain or branched alkanoyl, such as formyl, acetyl, propanoyl, butanoyl, 2-methylpropanoyl, pentanoyl, 2,2-dimethylpropanoyl, hexanoyl, heptanoyl, octanoyl, nonanoyl, decanoyl; alkoxycarbonyl, such as methoxycarbonyl, ethoxycarbonyl, t-butoxycarbonyl, t-pentyloxycarbonyl or heptyloxycarbonyl; cycloalkylcarbonyl such as cyclopropylcarbonyl cyclobutylcarbonyl, cyclopentylcarbonyl or cyclohexylcarbonyl; alkylsulfonyl, such as methylsulfonyl or ethylsulfonyl; alkoxysulfonyl, such as methoxysulfonyl or ethoxysulfonyl; heterocyclylcarbonyl; heterocyclylalkanoyl, such as pyrrolidinylacetyl, pyrrolidinylpropanoyl, pyrrolidinylbutanoyl, pyrrolidinylpentanoyl, pyrrolidinylhexanoyl or thiazolidinylacetyl; heterocyclylalkenoyl, such as heterocyclylpropenoyl, heterocyclylbutenoyl, heterocyclylpentenoyl or heterocyclylhexenoyl; or heterocyclylglyoxyloyl, such as, thiazolidinylglyoxyloyl or pyrrolidinylglyoxyloyl.
The term xe2x80x9cbiomoleculexe2x80x9d as used herein denotes biological molecules such as proteins, enzymes and other peptides, genetic material such as chromosomal material, genomic DNA, cDNA, mRNA, tRNA and other oligo- and polynucleotides. The term includes naturally occurring biological molecules in addition to fragments and recombinant derivatives thereof.
The divalent linking group Y may be saturated or mono-, di- or poly-unsaturated. Accordingly, the group Y may be any of the linking groups described above in which one or more of carbon-to-carbon single bonds is replaced by a double bond. For example the divalent linking group may further include alkenylene moieties such as butenylene; cycloalkenylene moieties such as 1-cyclohexenylene, alkenedioyl moieties such as fumaryl, maleyl, citraconyl and mesaconyl: heterocyclyl moieties such as pyrroline, imidazoline, pyrazoline and oxazoline.
Other suitable divalent linking groups include amino substituted groups such as glutamyl, aspartyl and asparaginyl, hydroxy substituted groups derived from glyceric acid, glycerol and pentaerythritol, and groups substituted with both hydroxy and amino such as threonyl.
Preferably linking group Y is selected from C1-7 alkylene, alkenylene, xe2x80x94(CH2CH2xe2x80x94O)pxe2x80x94, xe2x80x94(CH2CH2CH2xe2x80x94O)pxe2x80x94, 5 or 6 membered cycloalkylene or heterocyclyl, C1-7 alkenedioyl, C1-7 alkanedioyl, C1-7 alkylenedioxy, C1-7 alkylenedicarbonyl and groups of the formula xe2x80x94(R1)mxe2x80x94R2xe2x80x94(R3)nxe2x80x94, optionally substituted with one or two substituents selected from C1-5 alkyl, hydroxy, halo, amino, C1-5 alkyloxy and nitro; where R1 and R3 are selected from C1-5 alkylene, 5 or 6 membered cycloalkylene or heterocyclyl, xe2x80x94(CH2CH2xe2x80x94O)pxe2x80x94, xe2x80x94(CH2CH2CH2xe2x80x94O)pxe2x80x94, R2 is selected from xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94Sxe2x80x94Sxe2x80x94, xe2x80x94NRxe2x80x94, xe2x80x94NRC(O)Oxe2x80x94, xe2x80x94NRC(O)NRxe2x80x94, alkylenedioxyl, and xe2x80x94C(O)xe2x80x94, p is 1 to 8 and m, n and R are as defined above.
More preferably the linking group is selected from C1-5 alkylene, C1-5 alkenylene, xe2x80x94(CH2CH2xe2x80x94O)pxe2x80x94, C1-5 alkanedioyl, C1-5 alkylenedicarbonyl, xe2x80x94(C1-5 alkylene)mxe2x80x94R2xe2x80x94(C1-5 alkylene)nxe2x80x94, xe2x80x94(CH2CH2O)mxe2x80x94R2xe2x80x94(CH2CH2O)nxe2x80x94, optionally substituted with one or more substituents selected from C1-3 alkyl, hydroxy, halo, C1-3 alkyloxy; where R2 is selected from xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94Sxe2x80x94, xe2x80x94NRxe2x80x94 and NRC(O)NRxe2x80x94, where R is H or CH3, p is 1 to 4 and m and n are as defined above.
Most preferably the linking group Y is selected from xe2x80x94CH2xe2x80x94, xe2x80x94CH2xe2x80x94CH2xe2x80x94, xe2x80x94CH2xe2x80x94Oxe2x80x94CH2xe2x80x94, xe2x80x94CHOHCHOHxe2x80x94, xe2x80x94CH2xe2x80x94Sxe2x80x94Sxe2x80x94CH2xe2x80x94 and xe2x80x94(CH2)2NHC(O)NH(CH2)2xe2x80x94.
The crosslinking agents according to the present invention may be prepared by conventional methods. In one such method an appropriate substrate, i.e. a diamine, dialcohol, dithiol, aminoalcohol, thiolalcohol or thiolamine, is reacted with an equimolar amount of a reactive acryloyl or methacryloyl species, such as acryloyl chloride or methacryloyl chloride, in a suitable solvent such as chloroform or tetrahydrofuran, to form a monoacryloyl (or monomethacryloyl) intermediate. This intermediate may be isolated and purified before further reaction with an equivalent amount of the other reactive acryloyl or methacryloyl species, or the complete reaction may be carried out in two steps in a single pot. Reaction with the acryloyl species may be followed by reaction with the methacryloyl species or vice versa.
Where there is little or no reactivity differential between the reactive ends of the substrate, eg. for diamines, dialcohols and dithiols, it may be desirable to first protect one end of the substrate with an appropriate protecting group such as t-BOC. The other end can then be reacted with the reactive acryloyl or methacryloyl species to form a monoacryloyl (or monomethacryloyl) intermediate. Removal of the protecting group is then followed by reaction with the other reactive methacryloyl or acryloyl species. It is also possible to achieve reaction predominantly at one end of such a substrate by controlling the pH of the reaction mixture.
Some of the monoacryloyl and monomethacryloyl intermediates are novel compounds and represent a further aspect of the invention.
Preferred crosslinking agents useful in the present invention include the following: 
While not wishing to be limited by theory it is believed that the more reactive vinyl group will be preferentially incorporated into the polymer chain, for example the methacryloyl end of the crosslinking agent reacts first with the monomer to yield a polymer with pendant acryloyl units. In this way long linear chains of the monomer incorporating primarily methacryloyl groups of the crosslinking agent would be produced first, before crosslinking of the linear polymeric chains starts to occur. It is believed that this delayed crosslinking produces a gel having a microporous structure more suitable for the separation of biomolecules than known gels prepared from symmetrical crosslinking agents such as BIS.
Some of the bisamide, bithioester and amidethioester crosslinking agents according to the present invention are novel and accordingly in another aspect the invention there is provided a compound of formula: 
wherein X and Xxe2x80x2 are selected from xe2x80x94Sxe2x80x94 and xe2x80x94NRxe2x80x94, where R is H, alkyl or cycloalkyl, and
Y and Z are as defined above, provided that when X and Xxe2x80x2 are both xe2x80x94NRxe2x80x94, Y is not xe2x80x94CH2xe2x80x94 and does not include a quarternary ammonium group.
Some of the amide-ester and thioester ester crosslinking agents of the present invention are also novel and, accordingly, in another aspect of the invention there is provided a compound of formula: 
where one of X and Xxe2x80x2 is xe2x80x94Oxe2x80x94 and the other is selected from xe2x80x94Sxe2x80x94 and xe2x80x94NRxe2x80x94, where R is H, alkyl or cycloalkyl, and
Y and Z are as defined above; provided that Y is not C1-5 alkylene and does not include a quarternary ammonium group, and that when the other of X and Xxe2x80x2 is xe2x80x94NHxe2x80x94, Y is not methyleneoxy-2-hydroxypropylene.
In addition to being useful crosslinking agents in the preparation of crosslinked polymer gel, the novel compounds, in view of their ability to form cross linked polymer networks, may be used as precursors for novel polymeric materials, or they may be used in admixtures with other polymerisable entities to produce novel crosslinked polymeric compounds to form products such as optical lenses, dental cements, surface coatings, plastic films, heat resistant plastics and adhesives. These compounds also have potential as biologically active compounds (e.g. antitumour agents).
The gels of the present invention may be prepared by conventional methods. They may be prepared in a variety of polymer concentrations, depending on the sizes of the molecules to be separated. Polyacrylamide gels crosslinked with BIS are commonly used to separate DNA fragments less than 1 kb in length. For this purpose the gels are prepared having acrylamide concentrations in the range of about 3.5 to 20%. Gels having a concentration of 3.5% are useful for separating DNA fragments of about 100 to 1000 nucleotides while gels having an acrylamide concentration of about 20% are useful for separating fragments having from about 10 to 100 nucleotides.
Since the microporous structure of the gels according to the present invention is dependent on the particular crosslinking agent used, as well as the monomer concentration, the optimum monomer concentration for a particular biomolecule size range may differ somewhat from the optimum monomer concentrations known for acrylamide/BIS systems. The optimum concentration can be readily determined from standard trial runs.
Generally, however, the crosslinking agent can be employed in an amount of approximately 1 to 30 wt. %, preferably 2 to 10 wt. %, based on the total weight of the monomer and the crosslinking agent (% C). For the total gel concentration, monomers may be employed in approximately 1 to 50 wt %, preferably 1.5 to 20 wt %, based on total solution volume (% T).
The crosslinking polymerization reaction by which the novel gels of this invention are prepared is generally carried out in an aqueous medium and can be initiated by known initiators or polymerization catalysts. Suitable free radical-providing initiator systems are benzoyl peroxide, t-butylhydroperoxide, lauroyl peroxide, cumene hydroperoxide, tetralin peroxide, acetyl peroxide, caproyl peroxide, t-butylperbenzoate, t-butyldiperphthalate, methylethylketone peroxide, hydrogen peroxide-Fe2+-ascorbic acid, riboflavin-light, methylene blue-light, and various persulfate salts in conjunction with N,N,Nxe2x80x2,Nxe2x80x2-tetramethylethylenediamine (TEMED), diethylmethylaminediamine (DEMED), 3-dimethylaminopropionitrile (DMAPN) or similar reagents and ammonium persulfate-metabisulfite. Another class of free radical generating initiators are azocompounds such as azodiiosobutyronitrile, azodiisobutyramide, azobis (dimethylvaleronitrile), azobis (methylbutyronitrile), dimethyl, diethyl, or dibutylazobismethylvalerate. These and similar reagents contain a N,N double bond attached to aliphatic carbon atoms, at least one of which is tertiary. The amount and type of initiator is generally indicated by the nature and concentrations of the monomer and crosslinking agent used. The optimum amount of initiator is also affected by the presence of any accompanying impurities. Generally speaking, however, the initiator can be employed in the amount of approximately 0.3 to 5 wt. % based on the total amount of the monomer and crosslinking agent. The preferred initiator system is TEMED, DEMED or DMAPN and a persulfate salt.
Methods known in the art for utilizing polyacrylamide gels for determination of nucleotide sequences usually involve the preparation of the gels in given thicknesses, such as between glass plates, or plates of synthetic transparent material, to a thickness of approximately 3 mm. The gel may also be polymerized onto a support film. DNA samples labelled such as with 32P, 35S or fluorescent dyes are placed onto sample slots and electrophoresed. After electrophoresis (which generally occurs over a period of from 1 hour to a number of days) the gel is removed from the plates and autoradiography performed. In automated systems, fluorescent labelled nucleotides are monitored during the separation. Autoradiography requires 10 to 20 hours after which time films are studied to determine nucleotide sequence. The preparation of gels for autoradiography of 35S nucleotides requires immersion in 10% acetic acid to remove urea and handling of the gels with caution due to extreme fragility.
When proteins are to be separated by electrophoretic methods based on their size, an ionic detergent, such as sodium dodecyl sulfate (SDS) is generally added to the polyacrylamide gel, optionally in conjunction with other denaturants, to unfold the protein and provide a net negative charge. It is then possible to estimate molecular sizes from mobilities compared to known standards. In native electrophoresis where separation is made according to change and/or molecular weight, the polyacrylamide gels are generally used in combination with acidic, basic or neutral buffer systems in the absence of denaturing agents. Electrodes are positioned according to the predicted net charge of the sample at the pH used.
The gels according to the present invention may include conventional additives known to the art as required by the technique employed. These additives include detergents, such as SDS; denaturing agents, such as urea, N,Nxe2x80x2-dimethylformamide, n-propylalcohol, formamide, dimethyl formamide and glycine; high molecular weight polymers, such as polyvinyl alcohol, linear polyacrylamide, polyethylene glycols; and low molecular weight species such as glycerol and sucrose. The gels may also include a suitable buffer system.
A number of suitable buffer systems are disclosed in WO91/14489 the disclosure of which is incorporated herein by reference. These are shown below in Table I.
In addition to the analytical and preparative separation of biomolecules, the gels according to the present invention may be used to estimate molecular weights, and elucidate composition of complex mixtures. The gels may also be used in the sequencing of proteins and DNA, as polyelectrolytes, and may find use in environmental and quality control applications.
The performance of known gels has so far been limited by a number of factors including (a) restriction on pore size range available (thereby restricting the molecular weight range of fragments which can be separated), (b) the susceptibility of some gels to hydrolysis in slightly alkaline media, or to premature mechanical degradation and (c) high background staining caused by the amide linkages of BIS. An advantage of the present invention is the ability to control or modify gel formation to produce a gel having the desired characteristics (e.g. pore size, mechanical stability, alkaline stability, background staining etc.) by selecting an appropriate asymmetrical crosslinking agent.
In order to more clearly describe the invention reference will be made to the following examples and drawings which describe some preferred embodiments of the invention. However the particularity of the examples and drawings is not to be understood to supersede the generality of the preceding description.